Course Code: MBIO 306 Course Title: Immunology - 1

1. P R I M E A S I A U N I V E R S I T Y
a mission with a vision
Assignment : 1,2.
Topic:-
1, Antigen processing and presentation.
2. Antigen – Antibody Interaction,
Submitted to
Name: Sayeeda Be- Nozir
Designation: Lecturer
Department: Microbiology
Institute: Primeasia University
Submitted by
Name: Md Azizul Haque
Student ID: 193016031
Course Code: MBIO 306
Course Title: Immunology - 1
Department: Microbiology
Date of Submission: January 18, 2021

2. Antigen Processing and Presentation

3. Itruduction:-
In order to be capable of engaging the key elements of adaptive immunity (specificity, memory,
diversity, self/nonself discrimination), antigens have to be processed and presented to immune
cells. Antigen presentation is mediated by MHC class I molecules, and the class II molecules
found on the surface of antigen-presenting cells (APCs) and certain other cells. MHC class I and
class II molecules are similar in function: they deliver short peptides to the cell surface allowing
these peptides to be recognised by CD8+ (cytotoxic) and CD4+ (helper) T cells, respectively.
The difference is that the peptides originate from different sources – endogenous, or intracellular,
for MHC class I; and exogenous, or extracellular for MHC class II. There is also so called cross-
presentation in which exogenous antigens can be presented by MHC class I molecules.
Endogenous antigens can also be presented by MHC class II when they are degraded through
autophagy. ANTIGEN PROCESSING & PRESENTATION
Figure 1. The MHC class I antigen-presentation pathway
MHC class I presentation MHC class I molecules are expressed by all nucleated cells. MHC
class I molecules are assembled in the endoplasmic reticulum (ER) and consist of two types of
chain – a polymorphic heavy chain and a chain called β2- microglobulin. The heavy chain is
stabilised by the chaperone calnexin, prior to association with the β2-microglobulin. Without

4. peptides, these molecules are stabilised by chaperone proteins: calreticulin, Erp57, protein
disulfide isomerase (PDI) and tapasin. The complex of TAP, tapasin, MHC class I, ERp57 and
calreticulin is called the peptide-loading complex (PLC). Tapasin interacts with the transport
protein TAP (transporter associated with antigen presentation) which translocates peptides from
the cytoplasm into the ER. Prior to entering the ER, peptides are derived from the degradation of
proteins, which can be of viral- or self origin. Degradation of proteins is mediated by cytosolic-
and nuclear proteasomes, and the resulting peptides are translocated into the ER by means of
TAP. TAP translocates peptides of 8 –16 amino acids and they may require additional trimming
in the ER before binding to MHC class I molecules. This is possibly due to the presence of ER
aminopeptidase (ERAAP) associated with antigen processing. It should be noted that 30–70% of
proteins are immediately degraded after synthesis (they are called DRiPs – defective ribosomal
products, and they are the result of defective transcription or translation). This process allows
viral peptides to be presented very quickly – for example, influenza virus can be recognised by T
cells approximately 1.5 hours post-infection. When peptides bind to MHC class I molecules, the
chaperones are released and peptide–MHC class I complexes leave the ER for presentation at the
cell surface. In some cases, peptides fail to associate with MHC class I and they have to be
returned to the cytosol for degradation. Some MHC class I molecules never bind peptides and
they are also degraded by the ER-associated protein degradation (ERAD) system. There are
different proteasomes that generate peptides for MHC class-I presentation: 26S proteasome,
which is expressed by most cells; the immunoproteasome, which is expressed by many immune
cells; and the thymicspecific proteasome expressed by thymic epithelial cells.
Antigen presentation On the surface of a single cell, MHC class I molecules provide a readout of
the expression level of up to 10,000 proteins. This array is interpreted by cytotoxic T
lymphocytes and Natural Killer cells, allowing them to monitor the events inside the cell and
detect infection and tumorigenesis. MHC class I complexes at the cell surface may dissociate as
time passes and the heavy chain can be internalised. When MHC class I molecules are
internalised into the endosome, they enter the MHC class-II presentation pathway. Some of the
MHC class I molecules can be recycled and present endosomal peptides as a part of a process
which is called cross-presentation. The usual process of antigen presentation through the MHC I
molecule is based on an interaction between the Tcell receptor and a peptide bound to the MHC
class I molecule. There is also an interaction between the CD8+ molecule on the surface of the T
cell and non-peptide binding regions on the MHC class I molecule. Thus, peptide presented in
complex with MHC class I can only be recognised by CD8+ T cells. This interaction is a part of
socalled ‘three-signal activation model’, and actually represents the first signal. The next signal
is the interaction between CD80/86 on the APC and CD28 on the surface of the T cell, followed
by a third signal – the production of cytokines by the APC which fully activates the T cell to
provide a specific response. MHC class I polymorphism Human MHC class I molecules are
encoded by a series of genes – HLA-A, HLA-B and HLA-C (HLA stands for ‘Human Leukocyte

5. Antigen’, which is the human equivalent of MHC molecules found in most vertebrates). These
genes are highly polymorphic, which means that each individual has his/her own HLA allele set.
The consequences of these polymorphisms are differential susceptibilities to infection and
autoimmune diseases that may result from the high diversity of peptides that can bind to MHC
class I in different individuals. Also, MHC class I polymorphisms make it virtually impossible to
have a perfect tissue match between donor and recipient, and thus are responsible for graft
rejection. Antigen Processing and Presentation cont. MHC class II presentation MHC class II
molecules are expressed by APCs, such as dendritic cells (DC), macrophages and B cells (and,
under IFNγ stimuli, by mesenchymal stromal cells, fibroblasts and endothelial cells, as well as
by epithelial cells and enteric glial cells). MHC class II molecules bind to peptides that are
derived from proteins degraded in the endocytic pathway. MHC class II complexes consists of
αand β-chains that are assembled in the ER and are stabilised by invariant chain (Ii). The
complex of MHC class II and Ii is transported through the Golgi into a compartment which is
termed the MHC class II compartment (MIIC). Due to acidic pH, proteases cathepsin S and
cathepsin L are activated and digest Ii, leaving a residual class II-associated Ii peptide (CLIP) in
the peptide-binding groove of the MHC class II. Later, the CLIP is exchanged for an antigenic
peptide derived from a protein degraded in the endosomal pathway. This process requires the
chaperone HLA-DM, and, in the case of B cells, the HLA-DO molecule. MHC class II
molecules loaded with foreign peptide are then transported to the cell membrane to present their
cargo to CD4+ T cells. Thereafter, the process of antigen presentation by means of MHC class II
molecules basically follows the same pattern as for MHC class I presentation.
Figure 2. The MHC class II antigen-presentation pathway

6. As opposed to MHC class I, MHC class II molecules do not dissociate at the plasma membrane.
The mechanisms that control MHC class II degradation have not been established yet, but MHC
class II molecules can be ubiquitinised and then internalised in an endocytic pathway. MHC
class II polymorphism Like the MHC class I heavy chain, human MHC class II molecules are
encoded by three polymorphic genes: HLADR, HLA-DQ and HLA-DP. Different MHC class II
alleles can be used as genetic markers for several autoimmune diseases, possibly owing to the
peptides that they present.
Antigen-Antibody Interaction
The interactions between antigens and antibodies are known as antigen-antibody reactions. The
reactions are highly specific, and an antigen reacts only with antibodies produced by itself or
with closely related antigens. Antibodies recognize molecular shapes (epitopes) on antigens.
Generally, the better the fit of the epitope (in terms of geometry and chemical character) to the
antibody combining site, the more favorable the interactions that will be formed between the
antibody and antigen and the higher the affinity of the antibody for antigen. The affinity of the
antibody for the antigen is one of the most important factors in determining antibody efficacy in
vivo.
The antigen- antibody interaction is bimolecular irreversible association between antigen and
antibody. The association between antigen and antibody includes various non-covalent

7. interactions between epitope (antigenic determinant) and variable region (VH/VL) domain of
antibody. Chemical Bonds Responsible for the Antigen-Antibody Reaction The interaction
between the Ab-binding site and the epitope involves exclusively noncovalent bonds, in a similar
manner to that in which proteins bind to their cellular receptors, or enzymes bind to their
substrates. The binding is reversible and can be UG 4th Sem, Unit-IV prevented or dissociated
by high ionic strength or extreme pH. The following intermolecular forces are involved in Ag–
Ab binding: 1. Electrostatic bonds: This result from the attraction between oppositely charged
ionic groups of two protein side chains; for example, an ionized amino group (NH4 +) on a
lysine in the Ab, and an ionized carboxyl group (COO_) on an aspartate residue in the Ag. 2.
Hydrogen bonding: When the Ag and Ab are in very close proximity, relatively weak hydrogen
bonds can be formed between hydrophilic groups (e.g., OH and C=O, NH and C=O, and NH and
OH groups). 3. Hydrophobic interactions: Hydrophobic groups, such as the side chains of valine,
leucine, and phenylalanine, tend to associate due to Van der Waals bonding and coalesce in an
aqueous environment, excluding water molecules from their surroundings. As a consequence, the
distance between them decreases, enhancing the energies of attraction involved. This type of
interaction is estimated to contribute up to 50% of the total strength of the Ag–Ab bond. 4. Van
der Waals bonds: These forces depend upon interactions between the “electron clouds” that
surround the Ag and Ab molecules. The interaction has been compared to that which might exist
between alternating dipoles in two molecules, alternating in such a way that, at any given
moment, oppositely oriented dipoles will be present in closely apposed areas of the Ag and Ab
molecules. Each of these non-covalent interactions operates over very short distance (generally
about 1 Å) so, Ag-Ab interactions depends on very close fit between antigen and antibody.
Strength of Ag-Ab interaction: 1. Affinity: Affinity measures the strength of interaction between
an epitope and an antibody’s antigen binding site. It is defined by the same basic thermodynamic
principles that govern any reversible biomolecular interaction:
o KA = affinity constant
o [Ab] = molar concentration of unoccupied binding sites on the antibody
o [Ag] = molar concentration of unoccupied binding sites on the antigen
o [Ab-Ag] = molar concentration of the antibody-antigen complex
In other words, KA describes how much antibody-antigen complex exists at the point when equilibrium
is reached. The time taken for this to occur depends on rate of diffusion and is similar for every
antibody. However, high-affinity antibodies will bind a greater amount of antigen in a shorter period of

8. time than low-affinity antibodies. KA can therefore vary widely for antibodies from below 105 mol-1 to
above 1012 mol-1, and can be influenced by factors including pH, temperature and buffer composition.
Combined strength of total non-covalent interactions between single Ag- binding site of Ab and single
epitope is affinity of Ab for that epitope.
Low affinity Ab: Bind Ag weakly and dissociates readily.
High affinity Ab: Bind Ag tightly and remain bound longer.
2. Avidity: Antibodies and antigens are multivalent, meaning they possess more than one binding site.
The measure of the total binding strength of an antibody at every binding site is termed avidity. Avidity
is also known as the functional affinity.
Avidity is determined by three factors.
The binding affinity: The strength of the relationship at a singular binding site.
The valency: The total number of binding sites involved.
The structural arrangement: The structure of the antigen and antibody involved.
All antibodies are multivalent e.g. IgGs are bivalent and and IgMs are decavalent. The greater an
immunoglobulin’s valency (number of antigen binding sites), the greater the amount of antigen it can
bind. Similarly, antigens can demonstrate multivalency because they can bind to more than one
antibody. Multimeric interactions between an antibody and an antigen

9. help their stabilization.
A favorable structural arrangement of antibody and antigen can also lead to a
more stable antibody-antigen complex Strength of multiple interactions between multivalent Ab and Ag
is avidity.
Avidity is better measure of binding capacity of antibody than affinity. High avidity can compensate low
affinity.
3. Cross reactivity: Antibody elicited by one Ag can cross react with unrelated Ag if they share identical
epitope or have similar chemical properties.
Types of Ag-Ab reactions:
1. Agglutination
2. Precipitation
3. Complement Fixation
4. Enzyme linked Immunosorbent Assay
5. RadioImmuno Assay
6. Western Blotting
Agglutination
The interaction between antibody and a particulate antigen results in visible clumping called
agglutination. Antibodies that produce such reactions are called agglutinins. Better agglutination takes
place with IgM antibody than with IgG antibodies. Excess of an antibody also inhibits agglutination
reaction; this inhibition is called prozone phenomenon.
1. Agglutination is more sensitive than precipitation for the detection of antibodies.
2. Agglutination occurs optimally when antigens and antibodies react in equivalent proportions.
The prozone phenomenon may be seen when either an antibody or an antigen is in excess. Incomplete
or monovalent antibodies do not cause agglutination, though they combine with the antigen. They may
act as blocking antibodies, inhibiting agglutination by the complete antibody added subsequently.

10. Types of agglutination
1. Slide agglutination: Serotyping.
2. Tube agglutination: e.g. Widal test.
3. Indirect (passive agglutination): where soluble antigens are coated on vehicle particle e.g. latex
particle, RBCs.
Application of Agglutination reaction:
1. Cross-matching and grouping of blood.
2. Identification of Bacteria. E.g. Serotyping of Vibrio cholera, Serotyping of Salmonella Typhi and
Paratyphi.
3. Serological diagnosis of various diseases. E.g Rapid plasma regains (RPR) test for Syphilis,
Antistreptolysin O (ASO) test for rheumatic fever.
4. Detection of unknown antigen in various clinical specimens. E.g. detection of Vi antigen of Salmonella
Typhi in the urine.
Precipitation
It is a type of antigen-antibody reaction, in which the antigen occurs in a soluble form. When a soluble
antigen reacts with its specific antibody, at an optimum temperature and PH in the presence of
electrolyte antigen-antibody complex forms insoluble precipitate. This reaction is called a precipitation
reaction. A lattice is formed between the antigens and antibodies; in certain cases, it is visible as an
insoluble precipitate. Antibodies that aggregate soluble antigens are called precipitins.

11. The interaction of antibody with soluble antigen may cause the formation of insoluble lattice that will
precipitate out of solution. Formation of an antigen-antibody lattice depends on the valency of both the
antibody and antigen. The antibody must be bivalent; a precipitate will not form with monovalent Fab
fragments. The antigen must be bivalent or polyvalent; that is it must have at least two copies of same
epitope or different epitopes that react with different antibodies present in polyclonal sera. Antigen and
antibody must be in an appropriate concentration relative to each other.
1. Antigen access: Too much antigen prevents efficient crosslinking/lattice formation.
2. Antibody access: Too much antibody prevents efficient crosslinking/lattice formation.
3. Equivalent Antigen and Antibody: Maximum amount of lattice (Precipitate) is formed.
Application of Precipitation reaction:
1. Detection of unknown antibody to diagnose infection e.g. VDRL test for syphilis.
2. Standardization of toxins and antitoxins.
3. Identification of Bacteria e.g. Lancified grouping of streptococci.
4. Identification of bacterial component e.g Ascoli’s thermoprecipitin test for Bacillus anthracis.
Complement Fixation

12. Complement fixation is a method that demonstrates antibody presence in patient serum. Complement
fixation is a classic method for demonstrating the presence of antibody in patient serum. The
complement fixation test consists of two components. The first component is an indicator system that
uses combination of sheep red blood cells, complement-fixing antibody such as immunoglobulin G
produced against the sheep red blood cells and an exogenous source of complement usually guinea pig
serum. When these elements are mixed in optimum conditions, the anti binds on the surface of red
blood cells. Complement subsequently binds to this antigen -antibody complex formed and will cause
the red blood cells to lyse The second component is a known antigen and patient serum added to a
suspension of sheep red blood cells in addition to complement. These two components of the
complement fixation method are tested in sequence. Patient serum is first added to the known antigen,
and complement is added to the solution. If the serum contains antibody to the antigen, the resulting
antigen Sheep red blood cells and the anti not been bound by an antigen known antigens, it is available
to bind to the indicator system of sheep cells and anti sheep antibody. Lysis of the indicator sheep red
blood cells signifies both a lack of antibody in patient serum and a negative complement fixation test. If
the patient’s serum does contain a complement the lack of red blood cell lysis The complement pathwa
antigen-antibody complex and leads to cell lysis Complement Fixation Complement fixation is a method
that demonstrates antibody presence in patient Complement fixation is a classic method for
demonstrating the presence of antibody in patient serum. The complement fixation test consists of two
components. omponent is an indicator system that uses combination of sheep red blood fixing antibody
such as immunoglobulin G produced against the sheep red blood cells and an exogenous source of
complement usually guinea pig ts are mixed in optimum conditions, the anti-sheep antibody binds on
the surface of red blood cells. Complement subsequently binds to this antibody complex formed and will
cause the red blood cells to lyse. second component is a known antigen and patient serum added to a
suspension of sheep red blood cells in addition to complement. These two components of the
complement fixation method are tested in sequence. Patient serum is first added to the n, and
complement is added to the the serum contains antibody to the antigen, the resulting antigen-antibody
complexes will bind all of the complement. Sheep red blood cells and the anti-sheep antibody are then
added. If complement has n bound by an antigen-antibody complex formed from the patient serum and
known antigens, it is available to bind to the indicator system of sheep cells and anti sheep antibody.
Lysis of the indicator sheep red blood cells signifies both a lack of in patient serum and a negative
complement fixation test. If the patient’s serum does contain a complement-fixing antibody, a positive
result will be indicated by the lack of red blood cell lysis. pathway: Complement binds to antibody
complex and leads to cell lysis. Complement fixation is a method that demonstrates antibody presence
in patient Complement fixation is a classic method for demonstrating the presence of antibody in
patient serum. The complement fixation test consists of two components. antibody complexes will bind
all of the complement. sheep antibody are then added. If complement has antibody complex formed
from the patient serum and known antigens, it is available to bind to the indicator system of sheep cells
and anti- sheep antibody. Lysis of the indicator sheep red blood cells signifies both a lack of in patient
serum and a negative complement fixation test. If the patient’s fixing antibody,